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</head>  
<body>  
    <nav>  
        <h2>Content</h2>  
        <ul>  
            <li><a href="#overview">Overview</a></li>  
            <li><a href="#download">Download Full-length circRNA Data</a></li>  
            <li><a href="#detection">Detect circRNA from RNA-seq Data</a></li>  
            <li><a href="#reference">Construct Reference Library</a></li>  
            <li><a href="#completion">Complete Partial circRNA</a></li>  
            <li><a href="#integration">Integrate Blood circRNA Data</a></li>  
            <li><a href="#annotation">Annotate circRNA</a></li>  
        </ul>  
    </nav>  
    
    <main>  
        <section id="overview">  
            <h2>Overview</h2>  
            <p>This document describes the complete process of identifying, annotating, quantifying, and analyzing circular RNAs from RNA-seq data in human blood.</p><br> 

            <p> $ sh AQUARIUM_HB.sh detect --fastq1 SRR6450118_1.fastq.gz --fastq2 SRR6450118_2.fastq.gz --outdir SRR6450118 </p><br> 
            <p> $ sh AQUARIUM_HB.sh reference --stoutlist_path stout.list_path.txt --reference_file SRR6450118/ReferenceSet.txt </p><br> 
            <p> $ sh AQUARIUM_HB.sh reconstruct --cirireport_file SRR6450118/full/ciri.report --stoutlist_file SRR6450118/vis/stout.list --reference_file SRR6450118/ReferenceSet.txt --outputdir SRR6450118 </p><br> 
            <p> $ sh AQUARIUM_HB.sh quant --fastq1 SRR6450118_1.fastq.gz --fastq2 SRR6450118_2.fastq.gz --gtfdir SRR6450118 --quantdir SRR6450118/quant </p><br> 
            

        </section>  

        <section id="download">  
            <h2>1. Download Full-length circRNA Data</h2>  
            <p>Download full-length circRNAs from the following Nanopore sequencing datasets:</p>  
            <ul>  
                <li><a href="https://drive.google.com/file/d/1jjMEzCEEaUaUHrJZLME5O5_8fbsmYU6q/view?usp=drive_link">All full-length circRNAs in FLcircAS database</a></li><br>  

                <li><a href="https://drive.google.com/file/d/1oLk3MDw4kTZDzO7iA9nSNmDLoKqKxYSx/view?usp=drive_link">All full-length circRNAs in IsoCirc database</a></li>  
            </ul>  
        </section>  

        <section id="detection">  
            <h2>2. Detect circRNA from RNA-seq Data</h2>  
            <p>This step focuses on detecting circRNA from RNA-seq data using the downloaded sample dataset (Project ID: PRJNA722046).</p>  
            <p>You can execute detection using the following command:</p>  
            <div class="code-block">  
                <code>$ sh AQUARIUM_HB.sh detect --fastq1 SRR6450118_1.fastq.gz --fastq2 SRR6450118_2.fastq.gz --outdir SRR6450118 </code>  
            </div>   
        </section>  

        <section id="reference">  
            <h2>3. Construct Reference Library for Human Blood circRNA</h2>  
            <p>The goal of this step is to build a comprehensive reference library containing information from full-length circRNAs.</p>  
            <div class="code-block">  
            <code>$ sh AQUARIUM_HB.sh reference --stoutlist_path stout.list_path.txt --reference_file SRR6450118/ReferenceSet.txt </code>
            </div>     
            
            <h3>3.1 Integrate Full-length circRNA from CIRI Output</h3>  
            <p>Execute the following command to integrate the full-length circRNA obtained from CIRI output:</p>  
            <div class="code-block">  
                <code>$ sh AQUARIUM_HB.sh reconstruct --cirireport_file SRR6450118/ciri.report --stoutlist_file SRR6450118/stout.list --reference_file SRR6450118/ReferenceSet.txt --outputdir SRR6450118 </code>  
            </div>  
            <p>This command effectively combines circRNA data from multiple samples for further analysis.</p>  

            <h4>Input Data Example</h4>  
            <div class="data-example">  
PRJNA352396/SRR4888615/detection/full/vis/stout.list<br>  
PRJNA352396/SRR4888616/detection/full/vis/stout.list<br>  
PRJNA352396/SRR4888617/detection/full/vis/stout.list<br>  
PRJNA352396/SRR4888618/detection/full/vis/stout.list<br>  
PRJNA352396/SRR4888619/detection/full/vis/stout.list<br>  
PRJNA352396/SRR4888620/detection/full/vis/stout.list<br>  
PRJNA352396/SRR4888621/detection/full/vis/stout.list<br>  
PRJNA352396/SRR4888622/detection/full/vis/stout.list<br>  
PRJNA352396/SRR4888623/detection/full/vis/stout.list<br>  
PRJNA352396/SRR4888624/detection/full/vis/stout.list  
            </div>  

            <h4>Output Data Example</h4>  
            <div style="overflow-x: auto; margin: 10px 0;">  
                <table class="data-datatable">  
                    <thead>  
                        <tr>  
                            <th>chr</th>  
                            <th>bsj</th>  
                            <th>start</th>  
                            <th>end</th>  
                            <th>isoformID</th>  
                            <th>isoform_state</th>  
                            <th>strand</th>  
                            <th>exon_count</th>  
                            <th>exon_length</th>  
                            <th>exon_total_length</th>  
                        </tr>  
                    </thead>  
                    <tbody>  
                        <tr><td>5</td><td>5:73074742|73077493</td><td>73074742</td><td>73077493</td><td>chr5|73074742,73077338|73074853,73077493|+</td><td>Full</td><td>+</td><td>2</td><td>112,156</td><td>268</td></tr>  
                        <tr><td>X</td><td>X:1188454|1188569</td><td>1188454</td><td>1188569</td><td>chrX|1188454,1188460|1188459,1188569|+</td><td>Full</td><td>+</td><td>2</td><td>6,110</td><td>116</td></tr>  
                        <tr><td>10</td><td>10:5794885|5800705</td><td>5794885</td><td>5800705</td><td>chr10|5794885,5800598|5795019,5800705|-</td><td>Full</td><td>-</td><td>2</td><td>135,108</td><td>243</td></tr>  
                        <tr><td>2</td><td>2:112299849|112300029</td><td>112299849</td><td>112300029</td><td>chr2|112299849|112300029|+</td><td>Full</td><td>+</td><td>1</td><td>181</td><td>181</td></tr>  
                        <tr><td>19</td><td>19:20934024|20935047</td><td>20934024</td><td>20935047</td><td>chr19|20934024,20934949|20934150,20935047|+</td><td>Full</td><td>+</td><td>2</td><td>127,99</td><td>226</td></tr>  
                        <tr><td>12</td><td>12:75538109|75538245</td><td>75538109</td><td>75538245</td><td>chr12|75538109|75538245|+</td><td>Full</td><td>+</td><td>1</td><td>137</td><td>137</td></tr>  
                        <tr><td>4</td><td>4:185916779|185916890</td><td>185916779</td><td>185916890</td><td>chr4|185916779|185916890|-</td><td>Full</td><td>-</td><td>1</td><td>112</td><td>112</td></tr>  
                    </tbody>  
                </table>  
            </div>  

            <h3>3.2 Generate CircRNA Reference for Human Blood</h3>  
            <p>Use the following script to generate the final circRNA reference list:</p>  
            <div class="code-block">  
                <code>$ Rscript PIPELINE_make_reference.R --input stout.list.allsample.txt --output CircRNA_Reference.txt</code>  
            </div>  
            <p>The output file CircRNA_Reference.txt will contain circRNA information from FLcircAS, IsoCirc, and RNA-seq data.</p>  
        </section>  

        <section id="completion">  
            <h2>4. Complete Partial circRNA</h2>  
            <p>In this step, we aim to fill in the missing information of partial circRNAs to ensure data integrity.</p>  
            <p>Execute the following commands:</p>  
            <div class="code-block">  
                <code>$ sh AQUARIUM_HB.sh reconstruct --cirireport_file SRR6450118/ciri.report --stoutlist_file SRR6450118/stout.list --reference_file SRR6450118/ReferenceSet.txt --outputdir SRR6450118</code><br>  
            </div>  
            <p>These commands generate circRNA-related data corresponding to each sample, enhancing the accuracy and reliability of our dataset.</p>  
        </section>  

        <section id="integration">  
            <h2>5. Integrate Blood circRNA Data</h2>  
            <p>In this stage, we integrate extracted circRNA information with sample data. Run the following command:</p>  
            <div class="code-block">  
                <code>$ Rscript make.circRNAgtf_to_isoformID.R filepath_stout.list.txt</code>  
            </div>  
            <p>This step ensures that circRNA data from different sources can be effectively combined, laying a solid foundation for subsequent analysis.</p>  
        </section>  

        <section id="annotation">  
            <h2>6. Annotation of Human Blood Full-Length circRNAs</h2>  
            <p>Example informations of human blood full-length circRNAs are shown below, annotaion results can be made using below informations</p>  
            <div style="overflow-x: auto; margin: 10px 0;">  
                <table class="data-datatable">  
                    <thead>  
                        <tr>  
                            <th>chr</th>  
                            <th>start</th>  
                            <th>end</th>  
                            <th>strand</th>  
                            <th>bsj</th>  
                            <th>transcript_id</th>  
                            <th>isoform_state</th>  
                            <th>ReferenceType</th>  
                            <th>datasetname</th>  
                            <th>sampleID</th>  
                            <th>circRNA_type</th>  
                            <th>host_gene</th>  
                            <th>ensembl_id</th>  
                            <th>IsoformType</th>  
                            <th>total_length</th>  
                            <th>exon_count</th>  
                        </tr>  
                    </thead>  
                    <tbody>  
                        <tr><td>12</td><td>9096612</td><td>9096703</td><td>+</td><td>12:9096612|9096703</td><td>chr12|9096612|9096703|+</td><td>Full</td><td>Full</td><td>PRJNA722046</td><td>SRR14240793</td><td>intron</td><td>A2M</td><td>ENSG00000175899</td><td>full</td><td>92</td><td>1</td></tr>  
                        <tr><td>12</td><td>9108653</td><td>9109427</td><td>+</td><td>12:9108653|9109427</td><td>chr12|9108653,9109100,9109422|9108658,9109267,9109427|+</td><td>Full</td><td>Full</td><td>PRJNA722046</td><td>SRR14240736</td><td>intron</td><td>A2M</td><td>ENSG00000175899</td><td>full</td><td>180</td><td>3</td></tr>  
                        <tr><td>12</td><td>9108653</td><td>9109427</td><td>+</td><td>12:9108653|9109427</td><td>chr12|9108653,9109357|9108797,9109427|+</td><td>Full</td><td>breakinRef_gtf</td><td>PRJNA722046</td><td>SRR14240771</td><td>intron</td><td>A2M</td><td>ENSG00000175899</td><td>break</td><td>216</td><td>2</td></tr>  
                        <tr><td>12</td><td>53314298</td><td>53315782</td><td>-</td><td>12:53314298|53315782</td><td>chr12|53314298,53315094,53315335,53315727|53314441,53315140,53315426,53315782|-</td><td>Full</td><td>Full,FLcircAS_SKOV3</td><td>PRJNA722046</td><td>SRR14240737</td><td>exon</td><td>AAAS</td><td>ENSG00000094914</td><td>full</td><td>339</td><td>4</td></tr>  
                        <tr><td>15</td><td>67222978</td><td>67223079</td><td>-</td><td>15:67222978|67223079</td><td>chr15|67222978|67223079|-</td><td>Full</td><td>Full</td><td>PRJNA722046</td><td>SRR14240783</td><td>intron</td><td>AAGAB</td><td>ENSG00000103591</td><td>full</td><td>102</td><td>1</td></tr>  
                        <tr><td>15</td><td>67226901</td><td>67236820</td><td>-</td><td>15:67226901|67236820</td><td>chr15|67226901,67231814,67232446,67235979,67236408,67236815|67226950,67231897,67232837,67236068,67236504,67236820|-</td><td>Full</td><td>breakinRef_gtf</td><td>PRJNA722046</td><td>SRR14240732</td><td>exon</td><td>AAGAB</td><td>ENSG00000103591</td><td>break</td><td>719</td><td>6</td></tr>  
                    </tbody>  
                </table>  
            </div>  
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